(1) Summary of the Invention
The present invention relates to a sustainable chicken cell line infected with vaccine strains of Marek's disease virus (MDV). In particular, the present invention relates to cell lines infected with vaccine strains of turkey herpesvirus (HVT) and vaccine strains of serotype-2 MDV which can be used as live virus vaccine to protect poultry against Marek's disease, wherein the MDV resides in these cultures as a lytic or non-lytic infection, depending upon growing conditions.
(2) Background of the Invention
Marek's Disease
Marek's disease (MD), is the most common clinical neoplastic condition of any animal, including humans, on earth (H. G. Purchase, in "Marek's Diseases: Scientific Basis and Methods of Control: Clinical disease and its economic impact." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass., pp. 17-42. (1985)). MDV is a highly contagious lymphoproliferative herpesvirus. There are three MDV serotypes: the oncogenic serotype-1 (MDV-1) ; the nononcogenic serotype-2 (MDV-2) ; and the nononcogenic serotype-3, turkey herpesvirus (HVT) (B. W. Calnek and R. L. Witter, in "Diseases of Poultry: Marek's Disease" (B. W. Calnek et al., Eds.), Iowa State University Press, Ames, Iowa pp. 342-385, (1991)).
Replication of MDV and HVT is typical of other cell-associated herpesviruses and has been extensively reviewed (L. J. N. Ross, in "Marek's Diseases: Scientific Basis and Methods of Control: Molecular Biology of the Virus." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass., pp. 113-150 (1985)). The three general types of cell-virus interactions recognized are: productive infection, latent, and transforming. The sequence of events in MDV-1 infected birds leading to transformation include, 1) initial lytic growth in B-cells, 2) a latent phase involving infected T-cells, 3) a second round of lytic infection, coincident with permanent immunosuppression, and 4) oncogenic transformation (B. W. Calnek and R. L. Witter, in "Diseases of Poultry: Marek's Disease" (B. W. Calnek et al., Eds.), Iowa State University Press, Ames, Iowa, pp. 342-385 (1991)). The sequence of events in MDV-2 and HVT infected birds appears to be limited to an initial lytic phase followed by latency in non T-cells or latency without oncogenic transformation (B. W. Calnek and R. L. Witter, in "Diseases of Poultry: Marek's Disease" (B. W. Calnek et al., Eds.), Iowa State university Press, Ames, Iowa, pp. 342-385 (1991)).
Avian Cell Lines
The poultry industry has always recognized the need for continuous avian cell lines that could be used for producing Marek's disease vaccines and simplify development of recombinant MDV vectors for polyvalent vaccines. Although many avian cell lines have been developed (K. Nazerian, Avian Pathol. 16:527-544 (1987)), until the present invention, no cell line could substitute for chicken embryo fibroblast (CEF) cells in vaccine production. Previous cell lines failed because they were either derived from virally transformed cells or, if derived from chemically transformed cells, the cells produced tumors when inoculated into chickens, or the maximum titer of virus recoverable from the cell line was insufficient for commercial production. Therefore, poultry vaccine producers continue to use primary CEF cells for producing Marek's disease and other live and killed vaccines, an expensive and labor intensive process dependent on a continual source of specified pathogen free (SPF) eggs for preparing CEF.
Marek's Disease Vaccines
Marek's disease vaccines are the most widely used vaccines in the poultry industry. Since development of live virus Marek's disease vaccines in the late 1970's, losses to Marek's disease have been significantly reduced (B. W. Calnek and R. L. Witter, in "Diseases of Poultry: Marek's Disease" (B. W. Calnek et al., Eds.), Iowa State University Press, Ames, Iowa, pp. 342-385 (1991)). The most widely used Marek's disease vaccines are live HVT or a bivalent mixture of live HVT and the pathogenic serotype 2 MDV. The bivalent mixture of HVT and serotype 2 MDV synergistically affords greater protection against Marek's disease especially in those situations where HVT is not fully effective (R. L. Witter, Avian Pathol. 11:49-62 (1982); R. L. Witter and L. F. Lee, Avian pathol. 13:75-92 (1984); R. L. Witter, "Marek's Diseases:Scientific Basis and Methods of Control: Principles of Vaccination." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass. pp. 203-250 (1985)).
Marek's disease vaccines have achieved worldwide use (M. Pattison, "Marek's Diseases: Scientific Basis and Methods of Control: Control of Marek's disease by the poultry industry: practical considerations." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass., pp. 341-350 (1985)), even though production of these vaccines requires weekly preparation of CEF cells. Therefore, vaccine production is very dependent on a continuous and reliable supply of fertile eggs from specified pathogen free (SPF) flocks. SPF flocks are raised under special conditions and are regularly demonstrated to be free of avian pathogens (D. H. Thornton, "Marek's Diseases: Scientific Basis and Methods of Control: quality control and standardization of vaccines." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass., pp. 267-292 (1985)). Any disruption in the supply of fertile SPF eggs would disrupt production of MDV vaccines. A continuous cell line for MDV vaccine production would have great economic benefits for the worldwide poultry industry.
Current Marek's disease vaccines are either suspensions of infected CEF or cell-free virus suspensions made from sonicated CEF infected with vaccine strains of Marek's disease virus. Since there are no sustainable cell lines suitable for propagating MDV, the MDV vaccine industry uses primary CEF for production of vaccine virus (A. E. Churchill, "Marek's Diseases: Scientific Basis and Methods of Control: Production of vaccines." (L. N. Payne, Ed.), Martinus Nijoff Publishing, Boston, Mass., pp. 251-266 (1985)). Primary CEFs have a finite life span (approximately two to three weeks) and must be prepared every one or two weeks, increasing costs for producing MDV vaccines.
The number of passages in CEF cell culture is limited for MDV. Continual cultivation of all three MDV serotypes in CEF results in attenuation of serotype-1 MDV and for serotype-2 and 3, loss of protective efficacy against MDV. The attenuation of serotype-1 MDV in culture has been extensively investigated and has been correlated to an expansion in the het region of the viral genome. This expansion can be easily monitored by Southern analysis or by PCR. The reason for loss of protective efficacy for high passage serotype-2 and -3 MDV is not known.
H. Ogura and T. Fujiwara (Acta Med. Okayama 41:141-143 (1987)) established a cell line (CHCC-OU2) by chemically transforming chicken embryo cells. The cell line was fibroblastic in appearance, contact inhibited, and did not form colonies in agar. The authors showed that CHCC-OU2 cell lines were not malignantly transformed, did not produce endogenous avian retroviruses, but were capable of supporting replication of Newcastle disease virus and several subgroups of avian retroviruses. However, the authors did not extend their study to the replication of other avian viruses such as Marek's disease virus and infectious bursal disease virus.